Sulfonyl-Containing Boronate Caps for Optimization of Biological

Nov 29, 2017 - School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, Indiana 47907, United States. J. Med. Chem. , 201...
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Article Cite This: J. Med. Chem. 2018, 61, 319−328

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Sulfonyl-Containing Boronate Caps for Optimization of Biological Properties of 99mTc(III) Radiotracers [99mTcCl(CDO)(CDOH)2B‑R] (CDOH2 = Cyclohexanedione Dioxime) Zuo-Quan Zhao,†,§ Min Liu,‡,§ Wei Fang,*,† and Shuang Liu*,‡ †

Department of Nuclear Medicine, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences, and Peking Union Medical College, No.167 North Lishi Road, Xicheng District, Beijing 100037, China ‡ School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, Indiana 47907, United States S Supporting Information *

ABSTRACT: In this study, different boronate caps were used to optimize biodistribution properties of radiotracers [99mTcCl(CDO)(CDOH)2B-R] (1, R = 3S; 2, R = 3SP; 3, R = 3MS; 4, R = 3DMS; 5, R = 3MSB; 6, R = 3MMS; 7, R = 3MSA; 8, R = 3DMSA; 9, R = 4S; 10, R = 4MS; 11, R = 4MSB). Among the 11 new 99mTc radiotracers, 2 shows the most promising characteristics of an optimal heart imaging agent. Its initial heart uptake is close to that of 99mTc-Teboroxime, but its heart retention time is significantly longer. Its heart/liver, heart/lung, and heart/muscle ratios are also better than those of 99mTcTeboroxime. The SPECT image quality with 2 in SD rats is better than that with 99mTc-Teboroxime. The high initial heart uptake, long heart retention, and high heart/background ratios make 2 an excellent SPECT radiotracer for MPI.

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We have been interested in 99mTc(III) complexes [99mTcL(CDO)(CDOH)2B-R] (CDOH2 = cyclohexanedione dioxime; L = Cl, F, N3, and SCN; R = alkyl or aryl) as heart imaging agents, 11−14 because [ 99m TcCl(CDO)(CDOH) 2 B-CH 3 ] (99mTc-Teboroxime) has high first-pass extraction fraction15−19 and shows a linear relationship between its heart uptake and myocardial blood flow at 1−2 min postinjection (p.i.).20 Boronate caps are shown to have significant impact on biological properties of 99mTc(III) radiotracers [99mTcCl(CDO)(CDOH)2B-R].12,14 Among the radiotracers evaluated in our previous studies,11−14 99mTc-PAboroxime ([99mTcCl(CDO)(CDOH)2B-PA], PA = 1H-pyrazol-3-yl) and 99mTc5Fboroxime ([99mTcCl(CDO)(CDOH)2B-5F], 5F = 2-formylfuran-5-yl) show the most favorable biodistribution properties for heart imaging.12,14 However, they both have relatively high liver uptake.12,14 Therefore, there is a continuing need to minimize the liver uptake and maximize heart/liver ratios while maintaining the high heart uptake. As a continuation of our previous studies,11−14 we now present evaluation of novel 99mTc(III) complexes [99mTcCl(CDO)(CDOH)2B-R] (Figure 1, 1, R = 3S; 2, R = 3SP; 3, R = 3MS; 4, R = 3DMS; 5, R = 3MSB; 6, R = 3MMS; 7, R = 3MSA; 8, R = 3DMSA; 9, R = 4S; 10, R = 4MS; 11, R = 4MSB) as new radiotracers for heart imaging. Various sulfonyl-

INTRODUCTION

Coronary artery disease (CAD) arises from the gradual narrowing of coronary arteries and the decrease of blood flow due to buildup of cholesterol-containing deposits (plaque). Complete blockage of coronary arteries may cause myocardial infarction (commonly known as heart attack). Myocardial perfusion imaging (MPI) with radiotracers is a noninvasive technique to show the heart function (how well the heart muscle is pumping) and areas of perfusion defects (the areas of heart muscle without enough blood flow).1−6 In order to evaluate perfusion defects with accuracy, the radiotracer heart uptake must be proportion to regional blood flow.3−10 Precise measurement of the blood flow is of great clinical importance in identifying CAD, defining severity of disease, assessing myocardial viability, establishing the need for surgical intervention, and monitoring treatment efficacy. An ideal perfusion radiotracer should have the biodistribution properties controlled by blood flow rather than receptor binding or metabolism. It should also have a high initial heart uptake, a stable myocardial retention and the capability to track regional blood flow at 0−5 mL min−1 g−1 in a linear fashion. Stable myocardial retention is important to maintain this linear relationship. Rapid decrease of heart uptake often reduces the myocardial extraction and leads to the loss of linear relationship between radiotracer heart uptake and myocardial blood flow rate.7,10 © 2017 American Chemical Society

Received: September 27, 2017 Published: November 29, 2017 319

DOI: 10.1021/acs.jmedchem.7b01412 J. Med. Chem. 2018, 61, 319−328

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Figure 1. Structures of complexes [99mTcCl(CDO)(CDOH)2B-R] (1, R = 3S; 2, R = 3SP; 3, R = 3MS; 4, R = 3DMS; 5, R = 3MSB; 6, R = 3MMS; 7, R = 3MSA; 8, R = 3DMSA; 9, R = 4S; 10, R = 4MS; 11, R = 4MSB) under evaluation for their potential as radiotracers for heart imaging.

SI1). The HPLC retention times follow the general ranking order of 11 (15.5 min) > 5 (15.3 min) > 8 (13.3 min) > 7 (13.0 min) > 6 (12.8 min) > 1 (12.4 min) ∼ 9 (12.5 min) > 3 (11.5 min) ∼ 7 (11.5 min) ∼ 10 (11.5 min) > 2 (8.5 min). Longer HPLC retention time (Table 1) indicates that the radiotracer is more lipophilic under the conditions used in this study. Dynamic Planar Imaging. Dynamic planar imaging was performed in SD rats for 1−11. Their initial heart uptake and washout kinetics are the most important parameters in evaluation of new 99mTc radiotracers. Table 2 lists image

containing boronate caps are utilized to modify the biological properties of their corresponding 99mTc(III) radiotracers because of their structural diversity. The ultimate goal is to discover and develop an optimal 99mTc radiotracer with a high initial heart uptake, good heart/liver ratios, and a myocardial retention time substantially longer than that of 99mTcTerboroxime. The high initial heart uptake and long myocardial retention will help maintain the linear relationship between the radiotracer heart uptake and myocardial blood flow rate.20

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RESULTS Radiochemistry. All new radiotracers 1−11 are prepared according to Chart I. Heating at 100 °C is required in order to Chart I. Radiosynthesis of New 11

99m

Table 2. Image Quantification Data for 1−11 To Compare Their Heart Uptake and Washout Kineticsa radiotracer

Tc(III) Radiotracers 1−

99m

Tc-Teboroxime

1 2 3 4 5 6 7 8 9 10 11

initial heart uptake (%ID) 6.0 4.7 4.9 4.3 4.9 4.9 5.1 5.4 6.0 4.5 5.4 6.0

± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.7 1.0 0.7 0.7 0.6 0.7 0.8 0.6 0.7 0.8 0.3

AUC

R2

± ± ± ± ± ± ± ± ± ± ± ±

0.9920 0.9685 0.9609 0.9609 0.9685 0.9810 0.9601 0.9159 0.9733 0.9571 0.9355 0.9920

135 141 134 104 141 114 110 143 102 101 123 135

11 10 29 8 10 12 12 17 7 6 7 11

a

Their initial heart uptake is defined as the %ID heart uptake at 1 min p.i. Experimental data were obtained from 4−5 animals for each radiotracer. AUC values are calculated according to their respective biexponential equations.

complete radiosynthesis. Table 1 summarizes their radiochemical purity (RCP) data and HPLC retention times. Their RCP is 95−98% without postlabeling chromatographic purification. They are all stable in solution for >6 h (Figure

quantification data and the area under curve (AUC) values. Their myocardial washout curves are best fitted with the biexponential function, from which their 1 min heart uptake and AUC values are calculated. Among the 99mTc radiotracers evaluated in this study, 4 and 8 have the highest AUC values. Radiotracers 1, 2, and 9 show the 1 min heart uptake and AUC values that are higher than those of 99mTc-Teboroxime. It is worth noting that the heart radioactivity obtained from planar image quantification include the radioactivity in both blood pool and myocardium. Thus, biodistribution studies were performed to compare their in vivo distribution properties at 2 min p.i. in SD rats (200−225 g). Comparison of Biodistribution Data. Table SI1 in Supporting Information lists the selected 2 min biodistribution data for 1−4, 6, and 8−11. Figure 2 compares their 2 min uptake in the heart and liver. The 2 min heart values follow the general ranking order of 1 > 2 ∼ 3 ∼ 9 > 10 ∼ 11 > 6 > 7 > 8.

Table 1. HPLC Retention Times and RCP Data for 1−11 99m

Tc(III) complex (compd no.)

[99mTcCl(CDO)(CDOH)B-3S] (1) [99mTcCl(CDO)(CDOH)B-3SP] (2) [99mTcCl(CDO)(CDOH)B-3MS] (3) [99mTcCl(CDO)(CDOH)B-3DMS] (4) [99mTcCl(CDO)(CDOH)B-3MSB] (5) [99mTcCl(CDO)(CDOH)B-3MMS] (6) [99mTcCl(CDO)(CDOH)B-3MSA] (7) [99mTcCl(CDO)(CDOH)B-3DMSA] (8) [99mTcCl(CDO)(CDOH)B-4S] (9) [99mTcCl(CDO)(CDOH)B-4MS] (10) [99mTcCl(CDO)(CDOH)B-4MSB] (11)

HPLC retention time (min)

RCP (%)

12.4 8.5 11.5 13.0 15.3 12.8 11.5 13.2 12.5 11.5 15.5

>95 >95 >95 >95 >95 >95 >95 >95 >95 >95 >95 320

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Figure 2. Comparison of uptake values of 1−4, 6, and 8−11 in the heart (A) and liver (B) at 2 min p.i. in SD rats (n = 3−4). Higher heart uptake suggests a better radiotracer. Lower liver uptake indicates less background radioactivity.

The liver uptake follows the order of 8 ∼ 2 < 9 ∼ 1 < 10 ∼ 3 ∼ 6 ∼ 10 < 4. All new 99mTc radiotracers have low lung uptake. There is no clear relationship between the lipophilicity (Table 1) and heart uptake (Table SI1) of a specific radiotracer. However, more lipophilic radiotracers tend to have higher liver uptake values. Since 1, 2, and 9 have the heart/liver ratios close to or better than that of 99mTc-Teboroxime,11 they were selected for full-scale biodistribution studies to compare their heart retention times and pharmacokinetics from the blood and normal organs. Impact of Blood Radioactivity. Inspection of biodistribution data for new 99mTc radiotracers evaluated in SD rats (Table SI1) showed that radiotracers 1, 2, and 9 all have low radioactivity in the blood and their 2 min heart uptake is higher than that of 99mTc-Teboroxime. There is an inverse linear relationship between the 2 min heart uptake and level of blood activity (Figure 3A, y = −4.8551x + 5.2139; R2 = 0.6582). The largest deviation arises from 1. Injection of the radiotracer into the blood circulation exposes it to red blood cells (RBCs, the most abundant cellular component) and albumin (the most abundant protein component) prior to encountering cell membranes of the tissue or organ, in which tissue uptake and retention are a measure of physiology or pathology. Since the 99m Tc radiotracer taken up by myocardium is free, its availability for myocardial extraction depends largely on the extent to which the cellular and protein-bound radiotracer molecules become free during perfusion. The cellular and protein binding is the key factor controlling availability of 99mTc radiotracer for tissue uptake. Therefore, it is critical to evaluate the RBC and albumin-binding properties of new 99mTc(III) radiotracers. In this study, blood samples were drawn from the animals injected with 1−3 and 8−11 at 2 min p.i. The cellular components were easily separated from blood plasma by centrifugation. The cellular components and supernatant were collected separately. The collected supernatant was allowed to pass through a

Figure 3. (A) Linear relationship between 2 min heart uptake and blood radioactivity for 1−4, 6, and 8−11. The 2 min heart uptake and blood radioactivity values were reported as an average from biodistribution studies. Standard error bars were not shown for its clarity. (B) Direct comparison of the % of 1−3 and 8−11 binding to RBCs and plasma proteins. All experimental data were obtained from the blood samples used in biodistribution studies in SD rats (n = 3−4). (C) Linear relationship between 2 min heart uptake and % albumin binding for 1−3 and 8−11.

membrane-based filter. The results from γ-counting of the collected solid, filter, and filtrate show that 1−3 and 9 have 48− 58% of total blood radioactivity binding to RBCs (Figure 3B) and only 2−3% binding to albumin. The remaining 39−49% of blood radioactivity is most likely free radiotracer in the filtrate. Radiotracers 8, 10, and 11 have 29−55% of blood radioactivity binding to RBCs, 5−15% to albumin, and 30−63% remaining free in blood plasma (Figure 3B). Radiotracers 1, 2, 3, and 9 all have little albumin binding (2−3% of the total blood radioactivity) and show high heart uptake (Figure 2, 3.3−5.4 %ID/g). Radiotracers 8, 10, and 11 have more albumin-binding (5−15% of the total blood radioactivity) and show relatively low heart uptake (Figure 2, 0.7−2.2 %ID/g). There is a regressive linear relationship between the 2 min radiotracer 321

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heart uptake and its % albumin-binding (Figure 3C, y = −0.2738x + 4.4609; R2 = 0.735). Impact of Boronate Caps. The biodistribution data (Table SI1) show that the boronate caps have a profound impact on biodistribution properties of new 99mTc radiotracers. For example, 1 and 9 are positional isomers and share almost the same lipophilicity as evidenced by their identical HPLC retention times (Table 1). Even though 1 and 9 share similar biodistribution patterns in normal organs (Table SI1), their heart uptake values are significantly different (5.38 ± 0.19 % ID/g for 1 and 3.31 ± 0.70 %ID/g for 9). Similar trend is also observed with 3 and 10. Replacing the benzene ring in 1 with a pyridine ring results in 2, which is less lipophilic (Table 1) due to the pyridine-N atom, and its 2 min heart uptake is lower than 1. Radiotracer 2 has more blood radioactivity than 1 (Table SI1). The liver uptake of 2 is lower than that of 1. Replacing the CH3 group in 1 and 9 with the CH3NH moiety leads to 3 and 10, both of which have lower heart uptake and more liver radioactivity than 1 and 9 (Table SI1). Radiotracer 4 is more lipophilic than 3 due to the extra N−CH3 moiety and had higher liver uptake with lower heart uptake than 3 (Table SI1). Substitution of the CH 3 SO 2 moiety in 1 with a (CH3)2NHSO2NH functional group leads to the formation of 8, which has more blood radioactivity (1.03 ± 0.01 %ID/g) than 1 (0.33 ± 0.04 %ID/g) because of the capability of 3DMSA moiety to bind to cellular and protein components in the blood (Figure 3). As a result, 8 shows lower heart uptake (0.72 ± 0.03 %ID/g). Its liver uptake (2.36 ± 0.42 %ID/g) is much lower than that of 1 (3.80 ± 0.27 %ID/g). Myocardial Washout Kinetics. Tables SI2, SI3, and SI4 list the biodistribution data for 1, 2, and 9, respectively. These three radiotracers are of particular interest because of their high 2 min heart uptake (Figure 2). Figure 4A compares washout kinetics of 1, 2, 9, and 99mTc-Teboroxime from the heart over the 60 min period. The curves are best fitted with the biexponential equation for 1 (y = 1.03 + 2.27e−0.893X + 4.45e−0.067X; R2 = 0.9832), 2 (y = 0.95 + 1.47e−0.389X + 2.50e−0.029X; R2 = 0.9800), and 9 (y = 1.11 + 1.27e−0.893X + 3.45e−0.067X; R2 = 0.9735). All three new radiotracers show the heart uptake higher than that for 99mTc-Teboroxime over the 60 min study period. The AUC values for 1 (AUC = 136), 2 (AUC = 120), and 9 (AUC = 92) are significantly higher than those reported for 99mTc-Teboroxime (AUC = 71).14 The AUC value of 1 is >2× that for 99mTc-Teboroxime over the 60 min period. A larger AUC value indicates more radioactivity in the heart during a specific period. As a result, it takes ∼4 min for 2 and 9 and ∼8 min for 1 to approach the heart uptake value of 99m Tc-Teboroxime (Figure 4, ∼3.0 %ID/g) at 2 min p.i.11 Since 99m Tc-Teboroxime has a linear relationship between its myocardial uptake and the blood flow rate (0−5 mL min−1 g−1) at 1−2 min p.i.,20 it is reasonable to believe the time window to maintain the same linear uptake/flow relationship will be 4−5 min for 2 and 9 and 8−10 min for 1, which should be long enough to acquire high quality heart images using both the standard and CZT-based SPECT cameras. Therefore, 1, 2, and 9 have significant advantages over 99mTc-Teboroxime. This statement is further supported by the extremely promising results from SPECT studies (Figure 4B). Because of its fast myocardial washout, 99mTc-Teboroximine is only useful for SPECT imaging at 0−5 min p.i. In contrast, high quality images are acquired during any of the 5 min window over the first 20 min after administration of 1 and 2. Since 9 has faster myocardial washout than 1 and 2 (Figure 4A), only the first 10

Figure 4. (A) Comparison of myocardial washout kinetics between 1, 2, 9, and 99mTc-Teboroxime. All experimental data are from biodistribution studies in SD rats (n = 3−4). (B) Comparison of coronal views of SPECT images for the SD rats injected with 1, 2, 9, and 99mTc-Teboroxime, respectively, at 0−5, 5−10, 10−15, 15−20, 20−25, and 25−30 min p.i. These images are used to demonstrate the advantage of 1 and 2 over 99mTc-Teboroxime.

min is useful for high quality SPECT images with 9 (Figure SI3). Normal Organ Excretion Kinetics. Figure 5 compares the uptake values in background organs (blood, liver, lungs, and muscle) and heart/background ratios for 1, 2, 9, and 99mTcTeboroxime. In general, 1, 2, and 9 have similar blood radioactivity levels (Figure 6: 0.33−0.45 %ID/g) with 99mTcTeboroxime (0.49 %ID/g),14 but their heart/blood ratios were much better because of their higher heart uptake. Radiotracers 1, 2, and 9 also had lower uptake in the lung and muscle. Their heart/lung and heart/muscle ratios are significantly (p < 0.05) higher than those of 99mTc-Teboroxime (Figure 5). Among the three new 99mTc(III) radiotracers evaluated in biodistribution studies, 1 has the highest heart uptake, but its heart/liver ratios are similar to those of 99mTc-Teboroxime. In contrast, 2 has the heart/liver ratios close to those of 99mTc-Teboroxime at 15 min p.i. because of its higher heart uptake and faster liver clearance kinetics. Radiotracer 2 also shares very similar liver and lung uptake with 99mTcPAboroxime,12 but its heart uptake (1.0−3.7 %ID/g) is higher than that for 99mTc-PAboroxime (0.9−3.0 %ID/g) over the 60 min period.12 Radiotracer 2 exhibits the uptake values in the liver (0.9−3.8 %ID/g) and lungs (0.4−1.5 %ID/g) that are lower than those of 99mTc-5Fboroxime (liver, 2.5−4.5 %ID/g; lung, 1.0−2.1 %ID/g) over the 60 min study period.14 Radiotracers 1, 2, and 9 all have low uptake in the fat (∼0.3 %ID/g) and blood vessels (0.4−0.6 %ID/g) around the coronary arteries. This observation is consistent with the low radioactivity accumulation above the heart in the planar images 322

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Figure 5. Direct comparison of organ uptake and heart/background ratios between 1, 2, 9, and 99mTc-Teboroxime. Biodistribution data for 99mTcTeboroxime are obtained from our previous study.11 (#) p < 0.05, significantly different from the value for 99mTc-Teboroxime. (∗) p < 0.01, significantly different from the value for 99mTc-Teboroxime. An optimal radiotracer should have lower uptake in blood, liver, lungs, and muscle with the heart/background ratios better than those of 99mTc-Teboroxime.

not significant (Figure 4) over the first 30 min period. In contrast, 1 has more the liver radioactivity, which overlaps significantly with that in the heart at the bottom of inferior wall in the 3D and VLA (vertical long axis) images. Therefore, 2 has a significant advantage over 1 because of its low liver uptake and excellent heart/liver contrast. This conclusion is fully supported by the biodistribution data (Figure 5). It also has the advantage over 99mTc-PAboroxime and 99mTc-5Fboroxime reported in our previous studies,12,14 due to its lower uptake in liver and lungs (Tables SI1 and SI3). SPECT Imaging in Pigs. Figure 7 shows the selected SPECT images of the pig (∼25 kg) administered with 2 (∼75

(Figure SI2) for the SD rats administered with 1, 2, and 9, respectively. The high initial heart uptake (Figure 4) and high heart/background ratios (Figure 5) strongly suggest that 1, 2, and 9 are better radiotracers than 99mTc-Teboroxime for SPECT MPI. SPECT Imaging. Figure 6 shows SPECT images of the SD rats at 0−5 min after administration of 1, 2, and 9 and 99mTcTeboroxime, respectively, to demonstrate the superiority of new 99mTc(III) radiotracers over 99mTc-Teboroxime. 99mTcTeboroxime is used for comparison purposes. Radiotracer 2 shows the best image quality with lowest uptake in the liver, lungs and muscle. The interference from liver radioactivity is 323

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MBq or ∼2 mCi). The best imaging window is 0−5 min. Excellent heart images are also be acquired with 2 at 5−10 min p.i., during which it is impossible to acquire high quality SPECT images of the heart with 99mTc-Teboroxime (Figure 4B). Its liver uptake increases steadily and peaks at ∼15 min (Figure 7). The heart/liver ratio decreases rapidly during the first 10 min with the heart-to-liver ratios being 0.75−3.25. The average heart/liver ratio of 2 for the first 4 min is 2.15, which is close to that reported for 18F-BMS747158 (2-tert-butyl-4chloro-5-[4-(2-(18F)fluoroethoxymethyl)benzyloxy]-2H-pyridazin-3-1, heart/liver = 2.0 from PET quantification in pigs)21 and higher than that for 99mTc-Sestamibi (heart/liver of 3.0 mL min−1 g−1.3,20 As a result, its heart uptake significantly underestimates the changes in blood flow at 5.0 min after administration, which makes it difficult to accurately track the regional blood flow rate.23−26 With tremendous developments in the CZT-based SPECT cameras,27−37 the nuclear cardiology community has been calling for better perfusion radiotracers with longer heart retention time and/or improved biodistribution properties.38−40 Development of such a radiotracer remains the key for future success of nuclear cardiology. The sulfonyl-containing boronate caps are useful for optimization of the heart uptake and pharmacokinetics of 99m Tc radiotracers. Among the 99mTc radiotracers evaluated in SD rats, 1 has the initial heart uptake much higher than 99mTcTeboroxime over the 60 min period (Figure 4). Its initial heart uptake is also higher than that of 99mTc-PAboroxime and 99mTc5Fboroxime reported in our previous study.12,14 It takes ∼8 min for 1 to reach the 2 min heart uptake value of 99mTcTeboroxime (Figure 4, ∼3.0 %ID/g). It is highly conceivable that the time window for 1 to maintain the linear uptake/flow relationship is 8−10 min, which is much longer than that of 99m Tc-Teboroxime (∼2 min). However, its high liver uptake imposes significant challenge for its clinical acceptance. Radiotracer 2 also has the higher heart uptake than that of 99m Tc-Teboroxime over the 60 min period (Figure 4). It takes ∼4 min for 2 to approach the 2 min heart uptake value of 99m Tc-Teboroxime, suggesting that 2 may have a 4−5 min window to maintain the linear uptake/flow relationship. Radiotracer 2 shares very similar lung uptake with 99mTcPAboroxime, but its heart uptake (1.0−3.7 %ID/g) is higher as compared with those of 99mTc-PAboroxime (0.9−3.0 %ID/g) over the 60 min period.12 Radiotracer 2 and 99mTc-5Fboroxime share similar heart uptake. However, the uptake values of 2 in the liver (0.9−3.8 %ID/g) and lungs (0.4−1.5 %ID/g) are lower than those of 99mTc-5Fboroxime (liver, 2.5−4.5 %ID/g; lung, 1.0−2.1 %ID/g) over the 60 min period.14 Radiotracer 2 shows the best image quality (Figures 6 and 7). Considering all these factors, we believe that 2 is a better radiotracer than 99m Tc-Teboroxime and 1 for SPECT MPI.

Figure 6. SPECT and SPECT/CT images of the SD rats administered with 1 (n = 1), 2 (n = 3), 9 (n = 1), and 99mTc-Teboroxime (n = 1), respectively: SA = short axis; VLA = vertical long axis; HLA = horizontal long axis. These images were obtained at 0−5 min p.i. with the camera being focused in the heart region using a u-SPECT-II scanner. Anesthesia was induced using an air flow rate of 350 mL/min and ∼3.0% isoflurane. 99mTc-Teboroxime is used only for comparison purpose. SPECT/CT images are used to illustrate the relative orientation and intensity of radioactivity in the heart region. Only one animal is used for each radiotracer. Radiotracer 2 has little interference from the liver radioactivity because of its low liver uptake. However, the liver radioactivity interference is significant in the 3D and VLA views of heart for SD rats administered with 1 and 9.

Figure 7. Top: SPECT images of the pig (n = 1; 22.5 kg) administered with ∼75 MBq (∼2 mCi) of 2 at rest. SPECT images were acquired using the Discovery NM 530c SPECT camera (GE, USA) every 5 min over the first 30 min to illustrate the optimal imaging window. The liver uptake increased steadily and peaked at 15−20 min. Bottom: Heart/liver ratios obtained from SPECT images over the first 20 min (every minute) after injection. The heart/liver ratio decreases rapidly due to its fast heart washout, but it remains >1.0 over the first 6−7 min p.i.

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have low heart uptake. If the radiotracer [99mTcCl(CDO)(CDOH)2B-R] (e.g., radiotracer 2) has little RBC- and albumin-binding, its blood radioactivity will be low, more radioactivity will be available for myocardial extraction, and the radiotracer will have high heart uptake. The liver uptake of 2 is close to that of 99mTc-Teboroxime (Figure 3). Its liver radioactivity peaks at ∼15 min in pigs (Figure 7). The heart/liver ratio over the first 4 min is close to that of 18F-BMS747158 21 and higher than that for 99mTcSestamibi.22 High initial heart uptake and high heart/liver ratios are important because dynamic imaging is often performed for quantification of blood flow during the first 5 min p.i.21,22 Due to tremendous improvement in spatial resolution and sensitivity for CZT-based SPECT cameras (such as D-SPECT and the Discovery NM 530c),27−37 it is possible to complete a SPECT scan in 2−5 min using only 4−5 mCi of 99mTc radiotracer. The use of D-SPECT also makes it easier to minimize the overlap between the heart and liver radioactivity because the patient is placed at the upright position, and liver tends to drop more downward than heart.

Why are the heart uptake values from planar image quantification (Table 2) so different from those from biodistribution (Tables SI1−SI4)? The radioactivity from image quantification is accumulative over a specific period while the heart uptake from biodistribution is obtained at a specific point after radiotracer injection. The heart uptake values from planar images include parts of the radioactivity in blood-pool and normal organs. The heart uptake values can be readily corrected by deducting the radioactivity in normal organs. However, it is almost impossible to separate the bloodpool radioactivity from that in myocardium. This may explain why 1 and 8 shared similar AUC values (Table 2), despite the fact that the 2 min heart uptake value of 1 (5.38 ± 0.19 %ID/g) is much higher than that of 8 (0.72 ± 0.03 %ID/g). Another important question is why blood radioactivity has such an impact on the initial heart uptake of 99mTc radiotracers (Figure 3A). Radiotracer 2 has ∼58% of the total blood radioactivity binding to RBCs, 2−3% binding to the albumin, and 39% remaining free in blood plasma (Figure 3B). Similar cellular binding properties were also observed with 1, 3, 8, and 9 (Figure 5). Even though the heart localization mechanism remains unknown for 99mTc(III) radiotracers [99mTcCl(CDO)(CDOH)2B-R], it is certain that the RBC- and albumin-binding plays a significant role in their biodistribution properties (Figure 3C). This conclusion is consistent with the results from studies on interactions between 99mTc-Teboroxime and RBCs.41 The results from this study also show that 99mTc radiotracers with more blood radioactivity have a lower heart uptake. There is a regressive linear relationship between the 2 min heart uptake and blood radioactivity (Figure 3A) and % albumin-binding of 99mTc radiotracers (Figure 3C). The total blood volume is 15−16 mL (16−18 g) for the SD rats of 200 g.15,20,42,43 In contrast, their hearts weigh only 0.65−0.70 g. As a result, small changes in blood radioactivity may result in a big difference in radiotracer availability for myocardial extraction. This may explain why low blood radioactivity is so important to maintain the high heart uptake of cardiac radiotracers. The next question is how the boronate caps influence biodistribution properties of complexes [99mTcCl(CDO)(CDOH)2B-R] (Figure 4). One possible explanation is related to the stability of the Tc−Cl bond. It was reported that complexes [99mTcCl(CDO)(CDOH)2B-R] are more stable when the R group contains two or more carbon atoms.15,16,44 More stable Tc−Cl bond makes it difficult for [99mTcCl(CDO)(CDOH)2B-R] to convert into the corresponding “hydroxide” complex [99mTc(OH)(CDO)(CDOH)2 B-R], which has lower first-pass extraction and less heart uptake than [99mTcCl(CDO)(CDOH)2B-R].16 However, this hypothesis does not explain the fact that radiotracers 1 and 9 share almost the same composition and have so much difference in their heart uptake and myocardial retention time. The alternative explanation is most likely related to their capability for cellular and protein binding. This study clearly show that (1) the boronate caps have significant impact on the blood activity of [99mTcCl(CDO)(CDOH)2B-R] (Figure 3A), (2) radiotracers [99mTcCl(CDO)(CDOH)2B-R] bind to both RBCs and albumin (Figure 3B), and (3) there is a regressive linear relationship between the radiotracer heart uptake and % albumin-binding (Figure 3C). If the radiotracer [99mTcCl(CDO)(CDOH)2B-R] (e.g., 8) has a high RBC and albuminbinding capability, more radiotracer molecules will be bloodbound after injection. As a result, there will be less radioactivity for myocardial extraction and the radiotracer is expected to

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CONCLUSIONS

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EXPERIMENTAL METHODS

Among the new 99mTc(III) radiotracers evaluated in SD rats, 2 shows the most promising characteristics of a heart imaging agent. Its initial heart uptake is well comparable to that of 99m Tc-Teboroxime, but its heart retention time is significantly longer. The heart/blood, heart/lung, and heart/muscle ratios of 2 are much higher than those of 99mTc-Teboroxime. The SPECT image quality with 2 is much better than that with 99m Tc-Teboroxime at 0−5 min p.i. The high initial heart uptake, long myocardial retention, and high heart/background ratios make 2 an excellent candidate for future translation in clinics.

Materials. Chemicals (citric acid, γ-cyclodextrin, cyclohexanedione dioxime (CDOH2), diethylenetriaminepentaacetic acid (DTPA), 3(N,N-dimethylaminosulfonyl)aminophenylboronic acid (3DMSA-B(OH)2), 3-(N,N-dimethylsulfamoyl)phenylboronic acid (3DMS-B(OH) 2 ), 4-methoxy-3-(morpholinosulfonyl)phenylboronic acid (3MMS-B(OH)2), 3-(methylsulfonyl)aminobenzeneboronic acid (3MSA-B(OH)2), 3-[(methylamino)sulfonyl]benzeneboronic acid (3MS-B(OH)2), 4-[(methylamino)sulfonyl]benzeneboronic acid (4MS-B(OH)2), 3-(methylsulfonyl)phenylboronic acid (3S-B(OH)2), 4-(methylsulfonyl)phenylboronic acid (4S-B(OH) 2 ), 3(methylsulfonyl)pyridineboronic acid (3SP-B(OH)2), (3-(N-methylsulfamoyl))phenylboronic acid (3MS-B(OH)2), 3(morpholinosulfonyl)phenylboronic acid (3MSB-B(OH) 2 ), 4(morpholinosulfonyl)phenylboronic acid (4MSB-B(OH)2), sodium chloride, and SnCl2·2H2O) were purchased from Sigma/Aldrich (St. Louis, MO) or Matrix Scientific (Columbia, SC). Na99mTcO4 was obtained from Cardinal HealthCare (Chicago, IL). Radio-HPLC Method. The radio-HPLC method for analysis of 99m Tc radiotracers used an Agilent HP-1100 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a β-ram IN/US detector (Tampa, FL) and Zorbax C8 column (4.6 mm × 250 mm, 300 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 1 mL/min. The mobile phase was isocratic with 30% solvent A (10 mM NH4OAc buffer, pH = 6.8) and 70% solvent B (methanol) between 0 and 5 min, followed by a gradient from 70% solvent B at 5 min to and 90% solvent B at 15 min and isocratic mobile phase with 10% solvent A and 90% solvent B. The RCP was reported as the percentage of area for the expected radiometric peak on each radioHPLC chromatogram of each 99mTc(III) radiotracer. Radiosynthesis and Solution Stability. Radiotracers [99mTcCl(CDO)(CDOH)2B-R] were prepared using the kit formulation.11−14 325

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according to literature method.45 Each Amicon Centrifree (Beverly, MA) ultrafiltration device (30 000 Da NMWL) was loaded with 300− 600 μL of the collected blood plasma above. The Centrifree devices were immediately centrifuged at 12 000 rpm for 25−30 min. The filter and collected filtrate were countered on a γ-counter. The 99mTc radioactivity on the filter represents the percentage of protein-binding, and that in the filtrate indicates the percentage of free 99mTc radiotracer. SPECT Imaging Protocol in SD Rats. SPECT imaging was performed with the u-SPECT-II/CT scanner (Milabs, Utrecht, The Netherlands) equipped with a 1.0 mm multipinhole collimator. The SD rat was placed into a shielded chamber connected to an isoflurane anesthesia unit (Univentor, Zejtun, Malta). Anesthesia was induced using an air flow rate of 350 mL/min and ∼3.0% isoflurane, and maintained using an air flow of ∼250 mL/min with ∼2.5% isoflurane during image data acquisition (6 frames: 75 projections over 5 min per frame). The animal was administered with the 99mTc radiotracer (120−150 MBq) in 0.5 mLof saline containing ∼20% propylene glycol by tail vein injection. Rectangular scans in regions of interest (ROIs) from SPECT and CT were selected on the basis of orthogonal X-ray images provided by the CT. After SPECT acquisition, the animal was allowed to recover in a shielded cage. Image Reconstruction and Data Processing. SPECT reconstruction was performed using a POSEM (pixelated ordered subsets by expectation maximization) algorithm with 6 iterations and 16 subsets. CT data were reconstructed using a cone-beam filtered backprojection algorithm (NRecon version 1.6.3, Skyscan). After reconstruction, the SPECT and CT data were automatically coregistered according to the movement of the robotic stage and resampled to equivalent voxel sizes. Co-registered images were further rendered and visualized using the PMOD software (PMOD Technologies, Zurich, Switzerland). A 3D Guassian filter (1.2 mm fwhm) was applied to smooth noise, and the LUTs (look up tables) were adjusted for good visual contrast. The images were visualized as both orthogonal slices and maximum intensity projections. SPECT Imaging Protocol in Pigs. SPECT studies were performed on 1 and 2 in normal pigs (23−26 kg). The imaging protocol was approved by the Fu Wai Hospital Animal Care and Use Committee (Beijing, China). The rest of the SPECT scan was acquired in list mode data format on a Discovery NM 530c CZT SPECT camera (GE, USA) and followed by a CT scan on a GE Discovery 640 SPECT/CT camera. The animal was anesthetized with intravascular injection of 3% pentobarbital sodium (30 mg/kg). Additional sodium pentobarbital was used when needed. The animal was maintained with mechanical ventilation, monitored with electrocardiogram (ECG), and kept in the same position. Each animal was injected with ∼75 MBq of 99mTc radiotracer without saline flushing. List mode acquisitions were immediately started and continued for 30 min. The image data were processed to create dynamic raw data (6 frames; 5 min per frame) and reconstructed with a SPECT dedicated processing software, MyoFlowQ (BCBTI, Taiwan). Quantitative dynamic images were then analyzed to measure heart/liver ratio with ImageJ 1.49 (the National Institutes of Health, United States). Data and Statistical Analysis. Biodistribution data, T/B ratios, and imaging quantification data were reported as an average ± standard deviation based on the results from four to six SD rats at each time point. Comparison between two radiotracers was made using a one-way ANOVA test. The level of significance was set at p < 0.05.

Each vial contains 2 mg of CDOH2, 4−5 mg of boronic acid, 50−60 μg of SnCl2·2H2O, 9 mg of citric acid, 2 mg of DTPA, 50 mg of NaCl, and 20 mg of γ-cyclodextrin. To a lyophilized vial was added 1.0 mL of 99m TcO4− solution (370−1110 MBq). The vial was heated at 100 °C for ∼10 min. A sample of the resulting solution was diluted with saline containing ∼20% propylene glycol to 3.7 MBq/mL. The diluted solution was analyzed by radio-HPLC and ITLC. The RCP was 95− 98% with minimal [99mTc]colloid (24 h. Animals were anesthetized with intramuscular injection of a mixture of ketamine (80 mg/kg) and xylazine (19 mg/kg) before being used for biodistribution and planar imaging studies. Protocol for Dynamic Imaging in SD Rats. Dynamic imaging was performed in SD rats (n = 5, 200−250 g). Each animal was administered with the 99mTc radiotracer (40−75 MBq). After injection, the animal was immediately (within 5−10 s) placed prone on a single head mini γ-camera (Diagnostic Services Inc., NJ). Planar images were acquired every 30 s during the first 5 min, followed by images every 2 min at 6−30, 40, 50, and 60 min p.i. The imaging data were stored digitally in a 128 × 128 matrix. After planar imaging, the animals were returned to a lead-shielded cage. Planar images were analyzed by drawing regions of the heart (heart radioactivity) and whole body (injected radioactivity). The background was corrected by drawing the region right above heart. The results were expressed as the percentage of injected radioactivity (%ID). The myocardial washout kinetics was determined by curve fitting with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). The data were reported as an average ± standard deviation. Comparison between two radiotracers was made using a one-way ANOVA test. The level of significance was set at p < 0.05. Biodistribution Protocol. Fifteen SD rats (200−250 g) were divided randomly into five groups. Each animal was administered with 100−111 KBq of 99mTc radiotracer via the tail-vein injection. Three animals were sacrificed by sodium pentobarbital overdose (100−200 mg/kg) at 2, 5, 15, 30, and 60 min p.i. Blood was withdrawn from heart. Organs of interest (heart, brain, fat, intestines, kidneys, liver, lungs, muscle, spleen, and vessels around the artery) were harvested, rinsed with saline, dried with absorbent tissues, weighed, and counted on a PerkinElmer Wizard 1480 γ-counter (Shelton, CT). The organ uptake was reported as the percentage of injected dose (%ID) or percentage of injected dose per gram of organ tissue (%ID/g) from three SD rats in each group. Comparison between two 99mTc radiotracers was made using one-way ANOVA test (GraphPad Prism 5.0, San Diego, CA). The level of significance was set at p < 0.05. Cellular and Protein-Binding Assay. Blood samples were withdrawn (0.5−1.0 mL) from the heart at 2 min p.i. and were centrifuged at 12 000 rpm (Microfuge 22R centrifuge, Beckman Coulter, Indianapolis, IN) for 10−15 min. The solid (mainly RBCs) and supernatant were collected and countered on a PerkinElmer Wizard 1480 γ-counter (Shelton, CT). The percentage of RBCbinding was calculated as percentage of the total blood radioactivity. Protein binding of 99mTc radiotracers was evaluated by ultrafiltration

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01412. Tables listing biodistribution data for new 99mTc(III) radiotracers; figures showing HPLC chromatograms, planar images, and SPECT images (PDF) 326

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phenyl); 99m Tc-4MSBboroxime (11), [ 99m TcCl(CDO)(CDOH)2B-4MSB] (4-MSB = 3-(morpholinosulfonyl)phenyl)

Molecular formula strings (but not available for all new radiotracers) (CSV)

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AUTHOR INFORMATION

(1) Henneman, M. M.; Schuijf, J. D.; van der Wall, E. E.; Bax, J. J. Non-invasive anatomical and functional imaging for the detection of coronary artery disease. Br. Med. Bull. 2006, 79−80, 187−202. (2) Di Carli, M. F.; Hachamovitch, R. New technology for noninvasive evaluation of coronary artery disease. Circulation 2007, 115, 1464−1480. (3) Baggish, A. L.; Boucher, C. A. Radiopharmaceutical agents for myocardial perfusion imaging. Circulation 2008, 118, 1668−1674. (4) Slomka, P. J.; Patton, J. A.; Berman, D. S.; Germano, G. Advances in technical aspects of myocardial perfusion SPECT imaging. J. Nucl. Cardiol. 2009, 16, 255−276. (5) Salerno, M.; Beller, G. A. Noninvasive assessment of myocardial perfusion. Circ.: Cardiovasc. Imaging 2009, 2, 412−424. (6) Stirrup, J.; Wechalekar, K.; Maenhout, A.; Anagnostopoulos, C. Cardiac radionuclide imaging in stable coronary artery disease and acute coronary syndromes. Br. Med. Bull. 2009, 89, 63−78. (7) Slomka, P. J.; Berman, D. S.; Germano, G. Absolute myocardial blood flow quantification with SPECT/CT: Is it possible? J. Nucl. Cardiol. 2014, 21, 1092−1095. (8) Nekolla, S. G.; Rischpler, C.; Nakajima, K. Myocardial blood flow quantification with SPECT and conventional tracers: a critical appraisal. J. Nucl. Cardiol. 2014, 21, 1089−1091. (9) Henzlova, M.; Duvall, W. The future of SPECT MPI: Time and dose reduction. J. Nucl. Cardiol. 2011, 18, 580−587. (10) Klein, R.; Hung, G.; Wu, T.; Huang, W. S.; Li, D.; deKemp, R. A.; Hsu, B. Feasibility and operator variability of myocardial blood flow and reserve measurements with 99mTc-sestamibi quantitative dynamic SPECT/CT imaging. J. Nucl. Cardiol. 2014, 21, 1075−1088. (11) Zheng, Y.; Ji, S.; Tomaselli, E.; Ernest, C.; Freiji, T.; Liu, S. Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl, F, SCN and N3; CDOH2 = cyclohexanedione dioxime). Nucl. Med. Biol. 2014, 41, 813−824. (12) Yang, Y.; Zheng, Y.; Tomaselli, E.; Fang, W.; Liu, S. Impact of boronate-capping groups on biological characteristics of 99mTc(III) complexes [99mTcCl(CDO)(CDOH)2B-R] (CDOH2 = cyclohexanedione dioxime). Bioconjugate Chem. 2015, 26, 316−328. (13) Liu, M.; Zheng, Y.; Avcibasi, U.; Liu, S. Novel 99mTc(III)-azide complexes [99mTc(N3)(CDO)(CDOH)2B-R] (CDOH2 = cyclohexanedione dioxime) as potential radiotracers for heart imaging. Nucl. Med. Biol. 2016, 43, 732−741. (14) Liu, M.; Fang, W.; Liu, S. Novel 99mTc(III) complexes 99m [ TcCl(CDO)(CDOH)2B-R] (CDOH2 = cyclohexanedione dioxime) useful as radiotracers for heart imaging. Bioconjugate Chem. 2016, 27, 2770−2779. (15) Narra, R. K.; Nunn, A. D.; Kuczynski, B. L.; Feld, T.; Wedeking, P.; Eckelman, W. C. A neutral technetium-99m complex for myocardial imaging. J. Nucl. Med. 1989, 30, 1830−1837. (16) Rumsey, W. L.; Rosenspire, K. C.; Nunn, A. D. Myocardial extraction of teboroxime: effects of teboroxime interaction with blood. J. Nucl. Med. 1992, 33, 94−101. (17) Marshall, R. C.; Leidholdt, E. M., Jr.; Zhang, D. Y.; Barnett, C. A. The effect of flow on technetium-99m-teboroxime (SQ30217) and thallium-201 extraction and retention in rabbit heart. J. Nucl. Med. 1991, 32, 1979−1988. (18) Johnson, L. L. Myocardial perfusion imaging with technetium99m-teboroxime. J. Nucl. Med. 1994, 35, 689−692. (19) Iskandrian, A. S.; Heo, J.; Nguyen, T.; Mercuro, J. Myocardial imaging with Tc-99m teboroxime: technique and initial results. Am. Heart J. 1991, 121, 889−894. (20) Beanlands, R.; Muzik, O.; Nguyen, N.; Petry, N.; Schwaiger, M. The relationship between myocardial retention of technetium-99m teboroxime and myocardial blood flow. J. Am. Coll. Cardiol. 1992, 20, 712−719.

Corresponding Authors

*W.F.: e-mail, [email protected]. *S.L.: phone, 765-494-0236; fax, 765-496-1377; e-mail, [email protected]. ORCID

Shuang Liu: 0000-0001-8587-6471 Author Contributions §

Z-Q.Z. and M.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank Dr. Andy Schaber for his assistance in SPECT studies. This work was supported by Purdue University, Research Grant R21 EB017237-01 (S.L.) from the National Institute of Biomedical Imaging and Bioengineering (NIBIB), and Grant 81771872 from the National Nature Science Foundation of China (W.F.).

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REFERENCES

ABBREVIATIONS USED

General Terms

AUC, area under curve; CAD, coronary artery disease; CT, computed tomography; CZT, cadmium-zinc-telluride; DTPA, diethylenetriaminepentaacetic acid; ITLC, instant thin layer chromatography; MPI, myocardial perfusion imaging; RCP, radiochemical purity; SPECT, single photon emission computed tomography Known Radiotracers 18

F-BMS747158-02, 2-tert-butyl-4-chloro-5-[4-(2-( 18 F)fluoroethoxymethyl)benzyloxy]-2H-pyridazin-3-1; 99mTc-Teboroxime, [99mTcCl(CDO)(CDOH)2B-CH3] (CDOH2 = cyclohexanedione dioxime); 99mTc-PAboroxime, [99mTcCl(CDO)(CDOH)2B-PA] (PA = 1H-pyrazol-3-yl); 99mTc-5Fboroxime, [99mTcCl(CDO)(CDOH)2B-5F] (5F = 2-formylfuran5-yl); 99mTcN-NOET, [99mTcN(NOET)2] (NOET = Nethoxy,N-ethyl(dithiocarbamato); 99mTc-Sestamibi, [99mTc(MIBI)6]+ (MIBI = 2-methoxy-2-methylpropylisonitrile) New Radiotracers 99m Tc-3Sboroxime (1), [99mTcCl(CDO)(CDOH)2B-3S] (3S = 3-(methylsulfonyl)phenyl); 99mTc-3SPboroxime (2), [99mTcCl(CDO)(CDOH)2B-3SP] (3SP = 5-(methylsulfonyl)pyridin3yl); 99mTc-3MSboroxime (3), [99mTcCl(CDO)(CDOH)2B3MS] (3MS = 3-[(methylamino)sulfonyl]phenyl); 99mTc3DMSboroxime (4), [99mTcCl(CDO)(CDOH) 2B-3DMS] (3DMS = 3-(N,N-dimethylaminosulfonyl)aminophenyl); 99m Tc-3MSBboroxime (5), [ 99m TcCl(CDO)(CDOH) 2 B3MSB] (3MSB = 3-(morpholinosulfonyl)phenyl); 99mTc3MMSboroxime (6), [99mTcCl(CDO)(CDOH)2B-3MMS] (3MMS = 4-methoxy-3-(morpholinosulfonyl)phenyl); 99mTc3MSAboroxime (7), [99mTcCl(CDO)(CDOH) 2 B-3MSA] (3MSA = 3-(methylsulfonyl)aminophenyl); 99mTc-3DMSAboroxime (8), [99mTcCl(CDO)(CDOH)2B-3DMSA] (3DMSA = 3-(N,N-dimethylaminosulfonyl)aminophenyl); 99mTc-4Sboroxime (9), [ 99m TcCl(CDO)(CDOH) 2 B-4S] (4S = 4(methylsulfonyl)phenyl); 99mTc-4MSboroxime (10), [99mTcCl(CDO)(CDOH)2B-4MS] (4MS = 4-[(methylamino)sulfonyl]-

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DOI: 10.1021/acs.jmedchem.7b01412 J. Med. Chem. 2018, 61, 319−328